Objective To investigate the differences on lymphatic vessel density (LVD) among esophageal adenocarcinoma (EAC),esophageal squamous cell carcinoma (ESCC) and normal esophageal tissues,and analyze the clinical significance.Methods Twenty samples of EAC,24 samples of ESCC and 20 cases of normal esophageal tissues were obtained at the Affiliated Hospital of North Sichuan Medical College from January 2004 to January 2011.D2-40 was used for immunostaining of lymphatic vessels in EAC,and antibodies of D2-40 and Ki-67 were used together to detect proliferation of lymphatic vessels.The differences in the LVD among EAC,ESCC and normal esophageal tissues were analyzed.All data were analyzed using the analysis of variance or t test.Results D2-40 staining could identify the lymphatic vessels,and antibodies of D2-40 and Ki-67 could detect the proliferation of lymphatic vessels.The LVD of EAC,ESCC and normal esophageal tissues were (3.3 ± 1.7)/0.17 mm2,(4.6 ± 1.2)/0.17 mm2 and (3.8 ± 1.2)/0.17 mm2,respectively,with significant differences (F =5.44,P ＜0.05).The LVD of EAC was significantly lower than that of ESCC (t =3.074,P ＜ 0.05),while there was no significant difference in the LVD between the EAC and normal esophageal tissues (t =-1.022,P ＞ 0.05).There were significant differences in the LVD between the ESCC and normal esophageal tissues (t =2.395,P ＜ 0.05).There were significant differences in the LVD between EAC patients with deglutition discomfort and those with pain (t =3.092,P ＜ 0.05).There were significant differences in the LVD between EAC patients with course ＜6 months and those with course≥6 months (t =3.092,P ＜ 0.05).No statistical difference in clinicopathological parameters including gender,age,site of lesion,tumor diameter,pathological morphology,T stage,N stage,G stage,TNM clinical stage and lymph node metastasis were detected (t ＝ 1.130,1.020,F =0.082,t =0.799,F =0.692,t =0.694,1.820,0.353,0.969,0.969,P ＞ 0.05).Conclusions The LVD of EAC is lower than that of ESCC,but is similar to that of normal esophageal tissues.The LVD of EAC is correlated with the symptoms and course of patients.

Objective To investigate the levels of blood pressure, blood lipid, blood glucose, body mass index (BMI) as well as the epidemiological characteristics of dyslipidemia, hyperglycemia, hypertension and metabolic syndrome of Hangzhou citizens. Methods A total of 28 990 citizens in Hangzhou city who underwent health checkup were recruited in this study, including 10 179 males and 18 811 females. The average age of subjects was 65.05 years. Subjects were asked to complete questionnaires regarding personal characteristics. The physical examination emphasized measurement of height, waist and blood pressure. Blood samples were collected and subjected to serum glucose, TC, HDL-C, LDL-C and TG measurements. The values of the examinations was described as xˉ± s . The ratios were compared with chi-square test. The trend analysis was conducted by linear correlation test. Results The prevalence of hypertension and metabolic syndrome was 17.1% and 11.2% respectively. And the prevalence of overweight/obesity and hyperglycemia was 36.3%,8.1%,16.4%respectively. It was indicated that the men had higher prevalence of hyperglycemia, hypertension, metabolic syndrome and overweight compared with women. However, as to the dyslipidemia, men and women were totally different. Women were more prone to suffer from hypercholesterolemia and elevation of low-density lipoprotein cholesterol. Men were apt to suffer from hypertriglyceridemia and reduction of high-density lipoprotein cholesterol. Divided the subjects by age into three groups, it was suggested that the rates of dyslipidemia, hyperglycemia, hypertension and overweight/obesity increased along with the increment of age in women. Although the rates of metabolic disorders were higher in the group of men, the trend of increase with age was not as significant as in women. It could be seen in men that dyslipidemia and overweight/obesity were reduced with the increase of age. Conclusion The metabolic disorders in Hangzhou citizens showed their own characteristics. It is suggested that multiple strategies targeting at different sexes and age-groups should be formulated to prevent and control the occurrence of metabolic diseases.

BACKGROUNDTo evaluate the effects and toxicities of combination therapy of chemotherapy with hydroxycamptothecin (HCPT)+5-fluorouracil (5-FU)+cisplatin (DDP) and concurrent radiotherapy for advanced non-small cell lung cancer (NSCLC).

METHODSFrom September 1999 to May 2001, 31 patients with advanced NSCLC were enrolled in this study. They were given chemotherapy with HCPT 6mg/m² on days 1-5, 5-FU 300mg/m² on days 1-5, DDP 30mg/m² on days 1-3 and concurrent radiotherapy. The chemotherapy was repeated every 28 days as a cycle. Each patient should receive at least two cycles. The total dose of primary tumors varied up to DT 50-70 Gy/25-35f, and that of metastatic tumors up to DT 30-60 Gy/10-30f .

RESULTSAmong the 31 patients, 6 got complete response, 18 got partial response, 5 had stable disease and 2 had progressive disease, with an overall response rate of 77.4% (24/31). The median survival duration was 16.7 months. The 1- and 2- year survival rates were 54.7% and 30.2%, and 1- and 2- year local control rates were 61% and 40%, respectively. The main toxicities were marrow suppression and gastrointestinal symptoms.

CONCLUSIONSThe results suggest that HCPT-based chemotherapy combined with concurrent radiotherapy is effective for NSCLC and can improve the survival rate and life quality of the patients with lung cancer.

BACKGROUNDPrevious researches have proven that nm23-H1 gene is a tumor metastatic suppressor gene, however, it is still unknown about its exact molecular mechanisms. Site-directed mutagenesis can correctly change the base sequence and get mutant proteins. The aim of this study is to construct nm23-H1 mutant and EGFP fusion genes by site-directed mutagenesis, and to provide basement for studying the functional and biochemical mechanisms of nm23-H1 gene.

METHODSSite-directed mutagenesis of nm23-H1 gene was performed by modified QuikChange™ Site-directed Mutagenesis Kit method. Pure plasmid containing fusion gene of nm23-H1 and EGFP (PLXSN-nm23-H1-EGFP) was mini-prepared. Four pairs of mutagenic primers were synthesized in vitro and the desired five mutations, S44A, P96S, H118F, S120G and P96S-S120G were introduced into nm23-H1-EGFP fusion gene by PCR.

RESULTSFive nm23-H1 mutant and EGFP fusion genes, nm23-H1(S44A)-EGFP, nm23-H1(P96S)-EGFP, nm23-H1 (H118F)-EGFP, nm23-H1(S120G)-EGFP and nm23-H1(P96S-S120G)-EGFP, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant fusion genes were completely concordant with experiment design.

CONCLUSIONSFive nm23-H1 mutant and EGFP fusion genes are successfully constructed, which can be used in further studies. QuikChange™ site-directed mutagenesis is a simple, rapid and efficient method.

BACKGROUNDIt has been known that oncogenesis and development of lung can- cer is a complex process regulated many genes and involved in multistages. Recent studies have demonstrated that signal transduction abnormality may play a very important role in these procedures. The aim of this study is to investigate the influence of exogenous MEK1/2 pathway inhibitor U0126 on the expression and activity of extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.

METHODSThe expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell line L9981 (parent cell line with nm23-H1 gene hetero-deletion ) were detected by Western blot and immuno-precipitation technique after treating with different doses of U0126. The in vitro proliferative and invasive abilities of the lung cancer cell line were determined by MTT and modified Boyden chamber methods.

RESULTSThe ERK1/2 relative activity of L9981 gradually decreased accompanied with increase of U0126 doses, and a highly significant difference of phosphorylated ERK1/2 expression level existed among the different concentration groups of U0126 (P < 0.01), but no significant difference of total ERK1/2 expression was found among the different concentration groups of U0126 (P=0.387). After treatment with same concentration of U0126 for different time, the ERK1/2 relative activity of L9981 gradually decreased as the treatment time of U0126 prolonging, and a highly significant difference of phosphorylated ERK1/2 expression level was observed among different treatment time groups (P < 0.01). But no significant difference of total ERK1/2 expression level was found among different time groups (P=0.689). The inhibition of ERK1/2 pathway by MEK1/2 specific inhibitor U0126 targeting the ERK1/2 pathway of L9981 was dose- and time-dependent. After treatment with different concentration of U0126, the proliferation of L9981 gradually decreased accompanied with increase of U0126 concentration and a highly significant difference existed among different groups of U0126 concentration (P < 0.001). The in vitro invasion of L9981 gradually decreased accompanied with increase of U0126 concentration. No significant difference of in vitro invasion of L9981 was found among 0, 10 and 20μmol/l of U0126 (P > 0.05). A highly significant difference was observed when U0126 concentration increased to 40 and 60μmol/l compared with 0, 10 and 20μmol/l of U0126 (P < 0.001).

CONCLUSIONSThe inhibition of Ras-to-MAPK pathway by Ras-to-MAPK specific inhibitor U0126 targeting the Ras-to-MAPK pathway of the human high-metastatic large cell lung cancer cell line L9981 is dose-dependent and time-dependent. Suppressing or blocking of Ras-to-MAPK signal transduction pathway can reverse the invasive and metastatic phenotype of the human high-metastatic large cell lung cancer cell line L9981. These results suggest that the key kinase MEK1/2 of the ERK1/2 pathway may be a potent therapeutic target for human lung cancer.

BACKGROUNDTo construct recombinant adenoviral vector carrying Smad3D or Smad7 by a simplified means.

METHODSBased on AdEasy System, adenoviral backbone plasmid vector and shuttle vector carrying the gene of interest were transferred into E.coli BJ5183 by chemical transformation methods in special order. The homologous recombination was performed.

RESULTSRecombinant adenoviral vector pAd-Smad3D and pAd-Smad7 were constructed successfully, which were confirmed by restriction enzyme digesting.

CONCLUSIONSRecombinant adenoviral vector may be constructed quickly and efficiently in E.coli by sequential chemical transformation methods.

A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker's downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography (IMAC). This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA (without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.
Base Sequence ; Chromatography, Affinity ; methods ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutation ; NM23 Nucleoside Diphosphate Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

BACKGROUNDTo investigate gene diagnosis of micrometastasis in lymph nodes in patients with non-small cell lung cancer (NSCLC) and the feasibility of mucin 1 (MUC1) mRNA and cytokeratin 19 (CK19) mRNA as molecular marker to detect micrometastasis of lung cancer.

METHODSExpression of MUC1 mRNA and CK19 mRNA was detected in 119 lymph nodes taken from 31 patients with NSCLC, 35 lymph nodes from 10 patients with pulmonary benign diseases as controls by nested reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSIn the 119 lymph nodes from lung cancer patients, CK19 mRNA expression was detected in 66 lymph nodes (55.5%) and MUC1 mRNA expression was detected in 65 lymph nodes (54.5%) by RT-PCR. Neither CK19 mRNA nor MUC1 mRNA expression was observed in all the 35 lymph nodes in the benign pulmonary lesion group.

CONCLUSIONSThe results suggest that the detection of both MUC1 and CK19 mRNA might be helpful to diagnose NSCLC micrometastasis in lymph nodes. The establishment of this method may lead to an earlier diagnosis of metastasis for lung cancer.

BACKGROUNDTo establish a human large cell lung cancer cell line L9981-nm23-H1 transfected with wild type nm23-H1 gene.

METHODSpLXSN-nm23-H1-EGFP was constructed by gene clone technique, and L9981-nm23-H1 was established by infected virus of nm23-H1 gene. The DNA and protein expression of nm23-H1 were detected in the transgene large cell lung cancer cell line by PCR and Western blot.

RESULTSpLXSN-nm23-H1-EGFP was constructed successfully, and the nm23-H1 cDNA was inducted to L9981 cell. The protein of nm23-H1 could be detected in L9981-nm23-H1 cell.

CONCLUSIONSProtein of nm23-H1 is stably, continuously and high efficiently expressed in L9981-nm23-H1 cell.

BACKGROUNDTo study the regulation of nm23-H1 gene on the expression of integrins in human high-metastasis large cell lung cancer cell line L9981 by transfecting nm23-H1 into cell.

METHODSLipofect was used to transfect nm23-H1 into the L9981 cell line; semi-RT-PCR was used to detect the difference of integrin β1 and integrin β3 mRNA expressions between tranfected and non-transfected cell lines; flow cytometry was used to detect the difference of integrin β1 and integrin β3 protein expressions between transfected and non-transfected cell lines.

RESULTS(1) A transgenic cell line L9981-nm23-H1 was obtained, which could stably and effectively express nm23-H1. The mRNA expressions of integrin β1 and β3 in L9981-nm23-H1 cell line were remarkably lower than those in L9981 cell line. (2) The protein expressions of integrin β1 and β3 in L9981-nm23-H1 cell line were significantly lower than those in L9981 cell line too (P < 0.01).

CONCLUSIONSTransfection of nm23-H1 gene into the L9981 cell line can significantly down-regulate the mRNA and protein expressions of integrin β1 and β3 in this cell line, which indicates that nm23-H1 may reverse the metastasis potential of L9981 cell line through modulation of integrin expression.